Background/Rationale: HIV-1 infection and its prevention are of particular importance to the health of veterans, with 1 out of 250 veterans known to be HIV-1 infected. Prevention of HIV-1 was specifically mentioned as one of the 3 main goals in President Obama's National HIV/AIDS Strategy in 2010, as well as the Department of Veterans Affairs National HIV/AIDS Strategy Operational Plan in 2011. Our group has focused on a preventive vaccine for HIV-1 through study of potent antibodies directed at HIV-1. A limited number of persons infected with HIV-1 develop circulating plasma antibodies able to potently neutralize a wide variety of HIV-1 isolates representing different genetic subtypes. It is widely held that the characteristics and specificitis of such antibodies can be used to guide the development of HIV-1 vaccine candidates capable of eliciting protective humoral immunity in a target population. Current methods for study of these antibodies include isolation of antibodies from memory B cells, which may not always reflect the antibodies circulating in the blood. Our research has focused on the identification of characterization of the broad neutralizing antibodies directly from patient serum (without potential bias of selection), as neutralizing antibody characteristics seen in multiple individuals are more likely be raised in a broad population. We have noted particular biochemical signatures that point to a common dominant, acidic epitope that is targeted on the envelope of HIV. Objectives: The specific hypothesis of this proposal is that the broad HIV-1 neutralizing response in plasma is due to a shared acidic epitope on the gp120 envelope. The specific aims of the project are to 1) Directly isolate and sequence the antibodies responsible for the broad neutralization from the plasma of HIV-1 infected individuals, and 2) Map the corresponding epitope(s) of the broad neutralizing antibodies to test the hypothesis that a shared acidic epitope of gp120 is responsible for the broad HIV-1 neutralization response. Methods: We have identified 10 patients with broad neutralization, of which 3 will be studies in detail. The affinit purification (antigen, subclass, and light chain specific) and fractionation (free-flow isoelectric focusing) scheme can narrow the antibodies of interest, directly, from the plasma to individual species. These species will be tested for broad neutralization. Upon confirmation, the individual antibody bands will be sequenced de novo using both LC-MS and Edman degradation (N-terminal and internal sequencing). Epitope mapping of the new mAbs will be undertaken with Elisa, mutagenesis studies, and X-ray crystallography. Once the epitope is identified, Elisa and mutagenesis studies will be used to test the active fraction on the other 7 individuals to determine if this epitope is responsible for broad neutralization. Findings: Anticipated results wil be isolation of new mAbs, identification of common characteristics of broadly neutralizing abs, and the identification of a common acidic epitope that can direct broad HIV-1 neutralization. Status: We have completed enough work on the techniques described herein (as well as each alternative plan) to ensure that the aims are feasible and will be completed. Impact: If our hypothesis proves correct, then the results will be novel and directly applicable to the study of HIV vaccines. This study will provide a deeper understanding of possibilities of the broad HIV-1 neutralizing response in humans, and it will also have identified a naturally occurring epitope(s) that can be the target of potent cross-clade antibodies against HIV-1. This will have an impact on the designs of an HIV vaccine, which when developed will improve the health of veterans by preventing this life-long disease.